Thus, secretion of LEC-EVs by an impaired endothelium could be an alarm signal that instigate the self-defense of peripheral lymphatic vessels against an excessive inflammation. Indeed, in vitro experiments suggest that CCP obtained rapidly following the onset of symptoms does not damage the aHDLEC junctions as much as late-donated plasma. We identified a particular signature of CCP that would counteract the effects of an excessive inflammation on the lymphatic endothelium. Accordingly, an easy and efficient selection of convalescent plasma based on time of donation would be essential to promote the preservation of the lymphatic and immune system of infected patients.Convalescent plasma therapy (CPT) has gained significant attention since the onset of the coronavirus disease 2019 (COVID-19) pandemic. However, clinical trials designed to study the efficacy of CPT based on antibody concentrations were inconclusive. Lymphatic transport is at the interplay between the immune response and the resolution of inflammation from peripheral tissues, including the artery wall. As vascular complications are a key pathogenic mechanism in COVID-19, leading to inflammation and multiple organ failure, we believe that sustaining lymphatic vessel function should be considered to define optimal CPT. We herein sought to determine what specific COVID-19 convalescent plasma (CCP) characteristics should be considered to limit inflammation-driven lymphatic endothelial cells (LEC) dysfunction. CCP donated 16 to 100 days after the last day of symptoms was characterized and incubated on inflammation-elicited adult human dermal LEC (aHDLEC). Plasma analysis revealed that late donation correlates with higher concentration of circulating pro-inflammatory cytokines. Conversely, extracellular vesicles (EVs) derived from LEC are more abundant in early donated plasma (r = −0.413,  To date, an alarming number of people worldwide have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a coronavirus responsible for the disease commonly referred to as coronavirus disease 2019 (COVID-19) ,4,5,6. TEarly in the pandemic, COVID-19 convalescent plasma (CCP) was proposed as a potential therapeutic option for severe COVID-19. The approach consists of transfusing plasma from donors who recently recovered from the disease to hospitalized patients, and thus transfer humoral protection against the virus. The approach was very compelling, because it was readily available. Early in the pandemic, CCP was made available in the Unites States through the Food and Drugs Administration (FDA) Expanded Access program (EAP), and later through Emergency Use Access. Initial data from the EAP suggested that passive immunization was very effective, because patients receiving CCP with high anti-COVID-19 antibody titers were 30% less likely to die from the disease than those receiving CCP with low titers .The enthusiasm for CCP plummeted in winter 2021 after several major randomized clinical trials failed to show a benefit over standard of care ,9,10,11.Lymphatic transport is at the interplay between the immune response and the resolution of inflammation from peripheral tissues, including the artery wall . ConstitForty-five donors were recruited in this study and their anthropometric data are presented in p = 0.041)  (p = 0.035)  A. We the= 0.041) B. To measduction . In cont= 0.018) B. In oth= 0.035) C. Howevep = 0.007) (p = 0.002) (p = 0.010) (p = 0.013). In line with previous literature, the model could be further improved (r = 0.590) by including both symptom severity  and delay since the onset of symptoms , although the latter did not reach statistical significance.Casadevall et al. recommended the use of CCP from donors with severe symptoms, as they have more antibodies than donors with mild symptoms . Therefo= 0.007) A. So far= 0.002) B. Furthe= 0.010) C. In thep = 0.049) (p = 0.043)  D while a= 0.043) E. HereinSELE)  and of intercellular adhesion molecule 1 (ICAM1)  (p = 0.008) (CDH5) mRNA expression was also strongly correlated with the time of donation  (p = 0.013)  A,B. Give= 0.008) C. Immuno 0.0001) E. Moreov= 0.013) F. TherefGiven the association between a late donation and lymphatic endothelium dysfunction, we hypothesized that convalescent plasma donated at a later stage after the resolution of symptoms could contain more pro-inflammatory cytokines and mediators. Thus, we characterized the pro- and anti-inflammatory profile of cytokines contained in our convalescent plasma samples. We observed a positive correlation between the time of donation and the levels of pro-inflammatory mediators interleukin 1-beta (IL-1β), IL-13, IL-12p70, interferon gamma (IFNɣ), IL-17A, interferon gamma-induced protein 10 (IP-10), macrophage inflammatory protein-1 (MIP-1), IFNα and IL-1α A–I. We n+), total leucocytes (CD45+), LEC (CD45− podoplanin+), blood endothelial cells (CD45− CD62e+) and platelets ) in the plasma of COVID-19 convalescent donors  (+)  and delay since symptoms onset  were independent predictors of the concentration of CD45− podoplanin+ EVs in convalescent plasma. We herein demonstrate that although CD45− podoplanin+ EVs represents a minority of EVs in CCP, they are the only population to correlate with the time of donation, a characteristic believed to be beneficial for the lymphatic endothelium.EVs are small vesicles released from apoptotic or activated cells. They contain proteins, lipids, and mRNAs also present in their cell of origin. They are also used as prognostic markers for several pro-inflammatory diseases . In our t donors A–G. We oantified H. Intere= 0.004) I, wherea= 0.004) . In mult− podoplanin+ EVs and increased early apoptosis as well as decreased permeability, both markers of preservation of the integrity of lymphatic endothelial cells. Furthermore, unlike CD235a+, CD45+ and CD45− CLEC2+ EVs , CD45− podoplanin+ levels are positively correlated to the expression markers of the activation of LEC such as ICAM1 and vascular endothelial growth factor receptor 3 (VEGFR-3). Indeed, we observed a correlation between VEGFR-3 expression and markers of endothelial integrity  expression  and VEGFR expression , as well as with LEC necrosis-to-apoptosis ratio , MitoSOXTM Red-positivity ratio  and LEC permeability . Thus, the abundance of LEC-EVs in the plasma of convalescent donors could be a useful marker indicating that CCP promotes favorable lymphatic function with incubation.To distinguish the confounding effects of donation delay since symptoms, antibody titers, tested cytokines and LEC-EVs on lymphatic endothelial function, we performed multiple linear regression. LEC-EVs was the only variable to be independently associated with ICAM-1  and cells in the sub G0/G1 phase of the cell cycle    and immunoblot  (EVs production occurs during apoptosis or cell activation. We sought to determine the quantity of EVs secreted by the lymphatic endothelium in culture following CCP treatment. We quantified CD45 0.0001) A,B. Cellic cells . Likewis= 0.022) C, which = 0.039) D–F. ThusThe COVID-19 pandemic is affecting an exponentially growing number of people and is still a high-risk disease ,4,5. GivWe first assessed the association between the antibody concentrations and clinical parameters, since level of antibodies is the most used CCP selection parameter for clinical trials ,18. We oIn line with studies assessing the correlation between the severity of the disease and benefits of CPT ,24, we oSeveral clinical trials studied the time of administration of CCP to optimize remission ,22,45. TBonny et al. quantified cytokine and chemokine levels in CCP and demonstrated higher levels of IFNγ, IL-10, IL-15, IL-21, and MCP-1 compared to plasma of healthy donors . Since t− podoplanin+ EVs  in early donated plasma, whereas EVs levels from leucocytes, blood endothelial cells, platelets and erythrocytes remained unchanged depending on the time of donation.EVs are shown to be able to modulate and regulate the activation of the endothelium by increasing the expression of ICAM-1 . AdditioFLT4 and ICAM1, demonstrating a lymphatic endothelium activation following incubation of LEC-EVs-rich CCP.We then sought to determine the reason why LEC-EVs are more abundant in early donated plasma. We investigated the impact of LEC-EVs contained in the plasma on a lymphatic endothelium. LEC-EVs correlated with an increase in early apoptosis, but most importantly, a decrease in lymphatic endothelium permeability, as indicated by less ovalbumin diffusing through the endothelium. In several studies, EVs were identified as being used as defense mechanisms for sending alarm signals to target cells ,58. They0/G1 phenotype of the cell cycle. Finally, aHDLEC secreting the most amount of EVs were associated with less expression of VEGFR-3, emphasizing the altered state of the lymphatic endothelium. Indeed, in pro-inflammatory conditions, VEGFR-3 is deemed beneficial by promoting lymphangiogenesis and increasing lymphatic contraction capacity, thus limiting inflammation [Given the beneficial impact observed of LEC-EVs contained in the plasma on aHDLEC in culture, we hypothesized that these EVs were secreted by the dysfunctional and ill lymphatic endothelial cells of the donors in the attempt to preserve the surrounding lymphatic cells. To validate this, we examined the state of aHDLEC and correlated their condition to the quantity of EVs secreted in culture. The results observed seemed to corroborate our assumptions. Indeed, aHDLEC in culture secreting the highest amount of EVs were those approaching an inflammatory and apoptotic state, as characterized, respectively, by an increase in mitochondrial ROS production and a sub-Gammation ,29,34,59Plasma from 45 convalescent donors was collected by apheresis . Donors Primary adult human dermal lymphatic endothelial cells  were used in this study and called adult human lymphatic endothelial cells (aHDLEC) throughout the manuscript. The cells were cultured with EGM-MV-2  complete culture medium . Heparin  at a final concentration of 8 μg/mL was added to basal medium EBM-MV-2   or growth factors) to prevent clotting of the CCP. Antibiotics  at a final concentration of 1%, to ensure inhibition of bacterial growth, were also added to the medium. The cells were used at a passage between 4 and 6, when a confluence of at least 80% was reached. The experimental design of this study is illustrated in n = 26) were detached with accutase , centrifuged and transferred to a 96-well plate . Following centrifugation, aHDLEC were labeled with MitoSOXTM Red probe for 30 min at 37 °C. All samples were analyzed on a flow cytometer (BD FACSCelestaTM). The results obtained were analyzed using FlowJoTM version 10 software (Tree star). The gating strategy is shown in TM Red probe is described in 2O2, Sigma, cat. H1009) at 1 mM was used as a positive control for the experiment  were centrifuged, resuspended in cold Annexin V buffer 1X  and transferred to a 96-well plate. Following centrifugation, cells were labeled with Annexin V and propidium iodide (PI) for 15 min at room temperature (RT) and cold Annexin V buffer 1X was added. All samples were analyzed in flow cytometry (BD FACSCelestaTM). The results obtained were analyzed using FlowJoTM version 10 software. The gating strategy is shown in For the measurement of cell viability, treated aHDLEC (0/G1), replicative (S) and post-replicative and mitotic cells (G2/M)) [TM version 5.0 software . All CCP data (n = 14) were normalized to the control plasma. Information about the PI solution is described in Adult HDLEC were fixed in cold 70% ethanol, washed and stained with a propidium iodide (PI) solution (Biotium cat. #40017), which is fluorogenic and binds to nucleic acids in a stoichiometric manner to allow for the assessment of the proportion of cells in each phase of the cell cycle (pre-replicative (G (G2/M)) . The resAntibodies within convalescent plasma were determined by human anti-SARS-CoV-2 S RBD ELISA as previously described . BrieflyPro- and anti-inflammatory mediators within 41 convalescent plasmas were quantified according to the manufacturer’s protocol .n = 48), 10 μL of CCP or control plasma were added to 90 μL of Annexin V buffer 1X  supplemented with 10 μM D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) . The antibody mix including carboxyfluorescein succinimidyl ester (CFSE) to establish the presence of active esterases and antibodies binding major histocompatibility complex 1 (MHC-I), CD235a, CD45, C-type lectin-like type II (CLEC2), CD62e, and podoplanin with the concentrations described in For the staining of EVs in plasma (n = 26) of treated aHDLEC were quantified as described previously [TM) in which the 450/40 bandpass filter  was manually swapped after cytometer setting and tracking (CS and T) calibration with a 1 mm-thick magnetron sputtered 405/10 bandpass filter , referred to as V-SSC in this manuscript. Plots and histograms show all parameters in height (indicated as –H), as recommended for EV detection [TM software .Stained EVs in CCP and in the supernatant  was performed according to the manufacturer’s protocol .Measurement of cholesterol in CCP samples . The primers used are displayed in −ΔΔCT) and normalized to the housekeeping gene ACTB. All CCP samples were then normalized to the control plasma.Treated aHDLEC were harvested, suspended in RiboZol™ RNA Extraction Reagent and stored at −80 °C for at least 24 h. The RNA was extracted using the PureLink RNA Mini Kit extraction kit  according to the manufacturer’s protocol and quantified on a NanoDrop™ 1000 Spectrophotometer (ThermoFisher Scientific). Reverse transcription of RNA was performed using the High-Capacity cDNA Reverse Transcription Kit . Quantitative PCR was performed on the QuantStudio™ 3 (ThermoFisher Scientific) using 10 ng of complementary DNA (cDNA) mixed with Itaq™ Universal SYBRTM 488 Conjugate  for 7.5 min in the dark and fixed in PFA 2% for 25 min. Cells were then blocked in 2% Donkey Serum for 30 min at RT and incubated with anti-VE-Cadherin antibody  for 1 h at RT at a final dilution of 1/200. Secondary antibody Alexa FluorTM anti-rabbit 647  was incubated for 1 h at RT at a final dilution of 1/300. Adult HDLEC were then incubated with DAPI (1/250) for 30 min and visualized using an LSM 710 Confocal Microscope (Zeiss) equipped with a x63/1.4 oil dic objective. Images were analyzed using ImageJ software and relative intensity was obtained by normalization of CCP samples (n = 9) to the control plasma.Adult HDLEC were seeded in 8-well plates  at 30,000 cells per well until confluence was reached. Following treatment, aHDLEC were incubated with 10 ng/mL Wheat Germ Agglutinin, Alexa FluorTM 488 (50 ng/mL) (Invitrogen cat. 34781) in a phenol-free medium  for 20 min at RT followed by its recovery in the basolateral region. The medium containing the ovalbumin was transferred in a 96-well black flat plate and its fluorescence was read with the plate reader Synergy 2 (BioTek) with 485/20 nm of excitation and 528/20 nm of emission. All CCP samples (n = 12) were then normalized to the control plasma.Adult HDLEC were seeded in transwell inserts (Corning cat. 3470 and Greiner Bio-one cat. 662640) in a 24-well plate (vWR cat. 10861-700) and treated in the apical side. Inflamed and healthy aHDLEC were used as positive  and negative control (incubated with complete medium), respectively. Permeability was assessed by the addition of ovalbumin Alexa Fluorn = 24–26) was normalized with its respective non-phosphorylated or α/ß-tubulin expression followed by a normalization on the control plasma expression.Proteins were extracted from treated aHDLEC using ice-cold radioimmunoprecipitation (RIPA) assay buffer and the protein concentrations were determined using MicroBCA™ Protein Assay Kit . Protein samples were diluted in 4× Laemmli buffer, heated at 95 °C for 5 min and separated by electrophoresis on a 7.5% SDS-PAGE. Proteins were then transferred on a poly (vinylidene fluoride) (PVDF) membrane overnight at 4 °C. The membranes were blocked with 5% nonfat dry milk or 5% bovine serum albumin (BSA) in Tris-buffered saline  for 2 h at RT, then incubated with an anti-VE-Cadherin , anti-phospho-VE-Cadherin , anti-VEGFR-3  and anti-α/ß tubulin  overnight at 4 °C. The membranes were incubated with HRP-conjugated secondary anti-rabbit  and anti-mouse  antibodies for 1 h at RT. An ECL Blotting Substrates Kit  was used for detection. Each plasma sample .Normally distributed data are represented as mean and standard deviation (SD), whereas non-normally distributed data are represented as median and interquartile range. For continuous variables passing the Shapiro–Wilk normality test, a two-tailed Pearson’s correlation was performed. For variables that did not pass the normality test, a logarithmic or square-root transformation  was performed to obtain a homogeneous distribution. For the variables that still did not pass the normality test following the transformation, the initial values were used, but a non-parametric two-tailed Spearman’s correlation was performed. Finally, in In conclusion, we reported in this study a new and easily measurable parameter to take into consideration for the selection of CCP used in CPT. Our results demonstrate that an early donation of convalescent plasma could protect against lymphatic dysfunction, as it contains circulating LEC-EVs. We envision that the secretion of LEC-EVs by an impaired endothelium could be an alarm signal that instigates the self-defense of peripheral lymphatic vessels against an excessive inflammation. Contrariwise, a late donation could promote loss of endothelial integrity and increased permeability of the lymphatic endothelium. Dysfunctional lymphatic drainage could alter the clearance of immune cells and pro-inflammatory mediators, leading to the exacerbation of inflammation in infected COVID-19 patients. Accordingly, an easy and efficient selection of convalescent plasma based on time of donation would be essential to promote the preservation of the lymphatic and immune system of infected patients. Further clinical trials are needed to investigate whether early donations of CCP would be beneficial for the well-being of infected and hospitalized patients.In terms of limitations, the recruitment of patients occurred solely during the first half-year of the pandemic, which prevented a thorough and longitudinal follow up of donors, and excluded the effect of vaccine. A restricted amount of CCP donors is also another limitation of this study. The severity of the disease was based on self-assessment, and thus graded by donors themselves using a self-questionnaire. Clinical parameters that could have been insightful for the analysis of this study, such as body mass index, history of cardiac events and medications, were also unavailable. Finally, the total count of EVs produced by aHDLEC in vitro was calculated based on the total number found in the media minus the number of EVs contained in the plasma that was incubated on the lymphatic endothelial cell monolayer.


Focal sources (FS) are believed to be important triggers and a perpetuation mechanism for paroxysmal atrial fibrillation (AF). Detecting FS and determining AF sustainability in atrial tissue can help guide ablation targeting. We hypothesized that sustained rotors during FS-driven episodes indicate an arrhythmogenic substrate for sustained AF, and that non-invasive electrical recordings, like electrocardiograms (ECGs) or body surface potential maps (BSPMs), could be used to detect FS and AF sustainability. Computer simulations were performed on five bi-atrial geometries. FS were induced by pacing at cycle lengths of 120–270 ms from 32 atrial sites and four pulmonary veins. Self-sustained reentrant activities were also initiated around the same 32 atrial sites with inexcitable cores of radii of 0, 0.5 and 1 cm. FS fired for two seconds and then AF inducibility was tested by whether activation was sustained for another second. ECGs and BSPMs were simulated. Equivalent atrial sources were extracted using second-order blind source separation, and their cycle length, periodicity and contribution, were used as features for random forest classifiers. Longer rotor duration during FS-driven episodes indicates higher AF inducibility (area under ROC curve = 0.83). Our method had accuracy of 90.6±1.0% and 90.6±0.6% in detecting FS presence, and 93.1±0.6% and 94.2±1.2% in identifying AF sustainability, and 80.0±6.6% and 61.0±5.2% in determining the atrium of the focal site, from BSPMs and ECGs of five atria. The detection of FS presence and AF sustainability were insensitive to vest placement (±9.6%). On pre-operative BSPMs of 52 paroxysmal AF patients, patients classified with initiator-type FS on a single atrium resulted in improved two-to-three-year AF-free likelihoods . Detection of FS and arrhythmogenic substrate can be performed from ECGs and BSPMs, enabling non-invasive mapping towards mechanism-targeted AF treatment, and malignant ectopic beat detection with likely AF progression. Atrial fibrillation (AF) is the most common cardiac arrhythmia, with focal sources of excitation being an important mechanistic cause, especially in paroxysmal AF patients. The standard treatment for AF, pulmonary vein isolation, confines focal source activity within the veins, but may not eliminate the risk of AF since it leaves others untreated. We hypothesized that non-invasive electrical recordings, such as standard 12-lead electrocardiograms and 252-lead body surface potentials, could provide useful information for therapy planning. We generated a synthetic dataset of 2973 AF episodes, driven or initiated by focal and non-focal sources in patient-specific atrial geometries, to train three mechanism-inspired classifiers which determined the presence and location of focal sources, as well as the sustainability of AF in atria without a focal source present. We showed the mechanistic basis of using the pre-operative signals to predict AF susceptibility after focal ablation. Our classifiers were accurate, efficient and robust to inter-patient variability on the synthetic dataset. By using their pre-operative recordings to estimate AF mechanisms for 52 paroxysmal AF patients, our classifiers predicted a patient subgroup with improved two-to-three-year AF-free rates post-ablation, demonstrating the potential to guide mechanism-directed treatment based on non-invasive signals. Atrial fibrillation (AF) is the most common arrhythmia, and increases risks of stroke and heart failure. It is acknowledged that ectopic activities, such as focal sources (FS), can play an important role in AF, especially for paroxysmal AF patients . FS refeElectrical isolation or destruction of FS, both pulmonary vein (PV) and non-PV foci , 8, has Both ECGs and body surface potential maps (BSPMs) acquire electric potential signals from multiple leads over the torso, and are common non-invasive mapping tools to detect cardiac abnormality. BSPMs have been analyzed to assess organization of AF by looking at frequency components . They haTo extract FS from body surface potentials, we exploit their characteristics of high periodicity with a short cycle length (CL) . HoweverHypothesizing that sustained rotors during FS-driven episodes suggest an arrhythmogenic substrate which can be detected non-invasively with SO-BSS, the objective of this study was to non-invasively detect the presence of FS and an AF-susceptible substrate, as well as the location of the focal site if it exists, in order to suggest appropriate ablation targets. We did so using computer simulations of realistic AF episodes driven by FS and/or reentrant sources, and by modelling focal ablation by removing FS, to form digital twins in training the classifiers . AF induFS presence, FS location and AF sustainability as targets of AF ablation, as shown in the motivation box of initiator-type FS if AF was sustained after the focal ablation, and as a driver-type FS otherwise. AF after focal ablation for initiator-type FS was denoted as an FS-induced AF. A reentry driven AF episode was denoted as a reentrant AF. AF sustainability after removing the FS indicated AF inducibility from the FS on the atria.We identified the classification of We systematically changed the CL and the location of FS, and the concentration of Acetylcholine (ACh) ([ACh]), across five patient atria, and calculated the AF inducibility and the rotor duration for each case of CL, focal location and [ACh] on each patient. The means and 95% confidence intervals across five patients are shown in AF inducibility was the highest from the PV FS, followed by the left atrium (LA) FS. The highest AF inducibility was observed from FS with a CL of 180 ms from the PVs and from the LA, with means 0.95 and 0.88, respectively, and with a CL of 210 ms from the right atrium (RA), with mean 0.24. The CL range with non-zero AF inducibility was also larger for FS from the PVs and the LA than in the RA. ACh regulation increased AF inducibility from FS with CLs of 150–210 ms from the LA, and from FS with a CL of 150 ms from the PVs, but not for FS from the RA.For FS with the presence of sustained rotors, AF was more likely to be sustained than without. During FS-driven episodes, longer rotor duration was associated with higher AF inducibility, with an Area Under the Receiver Operating Characteristic Curve (AUROC) of 0.83. Rotor duration ≥ 100 ms occurred more frequently with initiator-type FS than driver-type FS . For FS with CLs of 150–180 ms from the LA and PVs, all cases where initiator-type FS had a longer rotor duration than driver-type FS were also associated with a high AF inducibility. The AF episodes induced by these initiator-type FS also had a similarly long rotor duration. The remaining 33.3% of initiator-type FS were driven by short-lived rotors or macro-reentries, mostly concentrated at a CL of 210 ms, regardless of the focal site. Over five patient meshes, FS at a CL of 210 ms showed a relatively high AF inducibility (mean 25.7%) and inter-patient variability (standard deviation (s.d.) ±18.5%), as shown in the second column of For visualization, selected frames of an initiator-type FS and a driver-type FS are shown in As can be seen from In our simulations, ACh regulation shortened the refractory period, opening conduction pathways in some directions around an otherwise blocked focal discharge, which also amounts to a decrease in the ERP around the focal site. Furthermore, ACh regulation increased the spatial action potential duration (APD) heterogeneity of the atrial tissue, which provided a substrate for fibrillatory waves with variable CLs. To illustrate the advantages of SO-BSS, we showed examples of sources decomposed from BSPMs of Patient 2 with SO-BSS, in initiator-type FS , driver-s1, s2, s4, s6, s7 and s10) that were synchronized (absolute difference ≤ 10 ms). The reentrant AF, however, had only three synchronized sources  with CLs similar to the dominant CL of 191 ms and its harmonics. This showed the necessity of combining multi-lead features to classify different AF mechanisms.However, through extracting multi-source CLs, different degrees of CL alignment between SO-BSS sources were shown across different mechanisms. As the driver-type FS contained only one type of periodic source, CLs of extracted sources were the most uniform in the driver-type FS in nine out of ten sources. The sources of an initiator-type FS were less synchronized, with six  with little change afterwards, likely due to the inherently small number of sources with distinctive CLs. For the FS location classifier, with a larger K, the accuracy dropped for BSPMs but increased for ECGs.To understand how different mapping systems and the number of sources K = 10 sources for SO-BSS with BSPMs as shown in s1-to-leads contribution used in this classifier is a spatial feature (assuming fixed locations of electrodes), which is by design sensitive to large variation of lead placements.To test the sensitivity to different vest placements, we varied lead positions by 10 and 20 degrees around the z axis via the center of mass, and 5 and 10 cm in the x, y, and z directions to test over the dataset of Patient 2, using K = 10 from BSPMs were visualized by being projected onto two-dimensional orthogonal bases using Principal Component Analysis (PCA) .Single-site repetitive pacing at specified locations was performed with CLs of 120, 150, …, 270 ms to model the clinically observed range of focal CLs ) = 960 reentrant AF simulations, with 549 sustaining for more than 3000 ms being preserved. These 1776 (driver-type FS) + 324 (initiator-type FS) + 324 (FS-induced AF) + 549 (reentrant AF) = 2973 segments were used as study data in the following steps.In total, there were 5 (patients) ×36  ×6 (CLs) ×2 ([ACh]) = 2160 FS cases, and a small number (60) of them were not launched as their universal atrial coordinates of foci referenced locations outside of the atrial tissue, as some coordinates pointed to cavities such as those of PVs. This left 2100 FS simulations, with 900 LA, 960 RA and 240 PV FS. The odds of inducing self-sustained AF for at least 1000 ms from FS under a specific condition, measured as the proportion of initiator-type FS, was termed π. To calculate sustained rotors, we kept rotors lasting for at least 100 ms, assuming the phase singularity point moves less than 2 mm within 1 ms. The total duration with at least one such rotor was also computed for analysis.In order to track rotors in these simulations, we calculated phase singularity points, i.e., the cores of the rotors. We first filtered the transmembrane voltage with a fourth-order 15 Hz low-pass Butterworth filter, removed the DC component with a 0 Hz notch filter, computed phase by applying the Hilbert transform, and then applied the Iyer-Gray method  which idLocal activation and repolarization times were marked by the upward-sloped intersection of -30 mV and downward-sloped intersection of -70 mV of the transmembrane voltage since the time of a focal discharge, respectively. We then calculated the APD by subtracting the repolarization time from the local activation time. ERP was estimated as the maximal focal CL whereby the FS encountered a complete conduction block at tissue surrounding the focus. Searched on the set of prescribed focal CLs, ERP was estimated between the maximal CL and the minimal CL where a complete conduction block did or did not occur around the pacing site, respectively.The proportional z-test was implemented by statsmodels Python package (version 0.11.1) . The stuK equivalent periodic atrial sources  and their CLs  from the body surface potentials, ranked by the source periodicity, using a SO-BSS method adapted from SOBI [We first extracted rom SOBI . The impτ was measured by an unbiased version of auto-correlation function (ACF), an empirical periodicity measurement for time series s(t) as a function of a time-lag τ [s(t).The periodicity of the extracted source over a time-lag me-lag τ :ACF(τ)=si was adopted from the time-lag within a set of time-lags TFS (see TFS) resulting in the maximal auto-correlation score of the source si,The estimated CL of each source k from k = 1, 2, …, K that satisfied ACFk(CLk) > CIk(CLk), where CIk(τ) denotes the range of 95% confidence interval for estimating ACFk(τ).To approximate the dominant rhythm in the atria, a dominant CL was taken by the first CLK = 1, around 0.3 second with K = 10, and around 2.4 seconds with K = 20.On a Intel Xeon Gold 6140 processor with 36 cores, the time to extract SO-BSS features on a 1000ms BSPM signal sampled at 1 kHz was around 0.2 second with FS presence, comprised of the union of initiator-type FS and driver-type FS as the positive class, and the rest being the negative; (2) AF sustainability, to predict if there is an arrhythmogenic substrate where AF can be sustained in the atria without focal discharges from the FS, where the positive class consists of the union of FS-induced AF, reentrant AF and initiator-type FS, and the negative class is driver-type FS; (3) FS location, trained and evaluated on the FS-driven episodes, to determine if the focal site is located on the RA (positive class), or from the LA or PVs (negative class). We also evaluated AF sustainability (with FS), which used the trained classifier from AF sustainability to distinguish between initiator-type FS (positive class) and driver-type FS (negative class).Three binary classifiers were formed: (1) K extracted atrial sources, 2 × K-dimensional characteristic features were used as input features for classification.An illustration of features for each classifier is shown in s1-to-leads contributions, specifying the contribution of the highest ranked periodic source s1 to the normalized signal of each lead. This encoded the spatial information of the first periodic source. As the SO-BSS sources are sign-agnostic, the signs of coefficients were fixed to one where a majority of coefficients were positive. The coefficients of all s1-to-leads vector were re-scaled to have the same maximum and minimum.The features of the FS location classifier were the The imbalanced sample sizes of each class may result in a classifier biased towards the majority class. To overcome this problem, we applied an oversampling algorithm SMOTE  to make K.To train and evaluate a model on the five simulated patient datasets, we adopted a nested leave-one-patient-out cross validation method, which reduced bias in estimating the true error of the classifier on small datasets . There wThe SO-BSS algorithm was implemented in Python (version 3.6). SMOTE was implemented by imbalanced-learn Python package (version 0.6.2) . The ranTo apply our trained classifier to predict patient’s AF mechanism  Step 4, The BSPM signals were acquired at a sampling rate of 1 kHz. QRS complex was extracted by the Pan-Tompkins algorithm . T wave To identify AF ablation targets from body surface potentials, we generated a large variety of AF episodes driven by FS and reentrant sources. We then determined the AF sustainability after removing FS. The FS and arrhythmogenic substrate were identified by a random forest classifier, with features of CL, periodicity as well as the signal contribution from the first source, extracted from body surface potentials using SO-BSS. From the computer models, we showed that longer rotor duration during FS episodes, which indicated an arrhythmogenic substrate for AF, was correlated with higher AF sustainability after FS removal with an AUROC of 0.83. With this biophysical basis, our classifiers were able to accurately predict the FS existence and location, and AF sustainability with robustness to both ECGs and BSPMs and inter-patient atrial variability. The mechanism prediction of FS presence and AF sustainability was also robust to extreme variations in vest placements. On 52 paroxysmal AF patients going through procedures based mainly on PV isolation, our classifiers predicted a patient subgroup achieving improved two-to-three-year AF-free rates , by estimating patient AF mechanisms using their pre-operative BSPMs. Our study demonstrates the potential for advancing the mechanism-targeted treatment in AF patients, as well as detecting malignant ectopic beats with likely AF progression in the general population.S1 Appendix(PDF)Click here for additional data file.S1 FigThe red stars mark the locations of the FS activated right before 2000 ms (at 1980 ms for (a) and at 1890 ms for (b)). The red arrows showed the movement of rotor wavefronts. The time was counted from the first pacing of the FS. A macro-reentry going through the coronary sinus can be seen from 2300–2700 ms on (a).(TIF)Click here for additional data file.S2 FigThe red arrows indicate the directions of wavefront propagation. The time was counted from the initiation of the AF. The rotational sources can be clearly seen.(TIF)Click here for additional data file.S3 FigThe red arrows indicated the directions of wavefront propagation. The red arrows indicate the directions of wavefront propagation. The time was counted from the initiation of AF. A local rotor appeared close to the base of the posterior left atrial wall where ACh islands were distributed. The potentials were also more heterogeneous with the introduction of ACh.(TIF)Click here for additional data file.S4 Figsi as ranked by their eigenvalues, respectively, with the bottom row showing the V1-lead ECG. The first column shows the signal amplitude over time. The second column shows the value of ACF(si) over time-lags up to 500 ms, ACFi, where red bars mark the CLi with labels of CLi and MaxACi outside and inside the bracket, or CLV1 and MaxACV1, and shading shows the 95% confidence interval of the ACF. The third column shows the FFT power spectral density PSDi or PSDV1, with the DF indicated by a red bar. a.u.: arbitrary unit.The first 10 rows show the top 10 sources (TIF)Click here for additional data file.S5 FigThe legend is the same as (TIF)Click here for additional data file.S6 FigThe legend is the same as (TIF)Click here for additional data file.S7 Figa.u.: arbitrary unit.(TIF)Click here for additional data file.S8 FigData points were collapsed where possible for better visualization.(TIF)Click here for additional data file.S9 FigαLA of 0–0.33, 0.33–0.67 and 0.67–1 and βLA of 0–1, respectively. Note that in (a), only the positions but not the sizes of ACh islands were indicated.The other areas without ACh islands are marked in gray. As mentioned in the text, fixed numbers of 60, 80, and 100 2mm-radius ACh islands (in yellow) were randomly distributed at areas of (TIF)Click here for additional data file.S10 Fighttps://doi.org/10.5281/zenodo.5105725.The other areas without the moderation of the initial states are marked in gray. To initiate a reentry on the LA, the LA body was split into 48 sectors, marked by 48 different colors in (b), around a non-excitable core. In this case, the non-excitable core has a radius of 0. The initial states were generated from an action potential after a complete depolarization of a cell, with selected states shown in (a). We provided example code to generate these initial states in (TIF)Click here for additional data file.S1 VideoThe white spheres below the right PVs mark the cores of sustained rotors.(MP4)Click here for additional data file.S2 VideoThere was no sustained rotor induced.(MP4)Click here for additional data file.S1 Table+ACh and −ACh denote with and without ACh, respectively. The blank entries represent either p-value > 0.05, or not applicable due to empty sample size.(PDF)Click here for additional data file.S2 TableK = 10 sources as an input parameter.Bold fonts mark the highest scores and the corresponding features. The parentheses mark signals used. SO-BSS took (PDF)Click here for additional data file.S3 TableAbsolute dominant CL difference refers to the absolute difference in the dominant CLs between the original setting and the variation, for which the mean and the standard deviation (s.d.) are shown.(PDF)Click here for additional data file.S4 TableNo.: Number of. s.d: standard deviation. Min: minimum. Max: maximum.(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


Anti-disease breeding is becoming the most promising solution to cyprinid herpesvirus-3 (CyHV-3) infection, the major threat to common carp aquaculture. Virus challenging studies suggested that a breeding strain of common carp developed resistance to CyHV-3 infection. This study illustrates the immune mechanisms involved in both sensitivity and anti-virus ability for CyHV3 infection in fish. An integrative analysis of the protein-coding genes and long non-coding RNAs (lncRNAs) using transcriptomic data was performed. Tissues from the head kidney of common carp were extracted at days 0  and 7 after CyHV-3 infection (the survivors) and used to analyze the transcriptome through both Illumina and PacBio sequencing. Following analysis of the GO terms and KEGG pathways involved, the immune-related terms and pathways were merged. To dig out details on the immune aspect, the DEGs were filtered using the current common carp immune gene library. Immune gene categories and their corresponding genes in different comparison groups were revealed. Also, the immunological Gene Ontology terms for lncRNA modulation were retained. The weighted gene co-expression network analysis was used to reveal the regulation of immune genes by lncRNA. The results demonstrated that the breeding carp strain develops a marked resistance to CyHV-3 infection through a specific innate immune mechanism. The featured biological processes were autophagy, phagocytosis, cytotoxicity, and virus blockage by lectins and MUC3. Moreover, the immune-suppressive signals, such as suppression of IL21R on STAT3, PI3K mediated inhibition of inflammation by dopamine upon infection, as well as the inhibition of NLRC3 on STING during a steady state. Possible susceptible factors for CyHV-3, such as ITGB1, TLR18, and CCL4, were also revealed from the non-breeding strain. The results of this study also suggested that Nramp and PAI regulated by LncRNA could facilitate virus infection and proliferation for infected cells respectively, while T cell leukemia homeobox 3 (TLX3), as well as galectin 3 function by lncRNA, may play a role in the resistance mechanism. Therefore, immune factors that are immunogenetically insensitive or susceptible to CyHV-3 infection have been revealed. Cyprinid herpesvirus-3 (CyHV-3) infection is a major threat to common carp aquaculture , leading+ T cells was also observed , hypoxia inducible factor 1 subunit alpha (hif1a), galectin-8 (LGALS8), rootletin, and paladin, have also been related to resistance against CyHV-3  during the CyHV-3 challenge, using the strategy shown in All procedures involving animals in this study were conducted according to the guidelines for the care and use of laboratory animals of Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences . The studies involving animals were reviewed and approved by the Committee for the Welfare and Ethics of Laboratory Animals of Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences.4 used in this study were obtained from Kuandian Research Base of Heilongjiang River Fisheries Research Institute . The breeding strain was the fourth generation after the selection of resistance to CyHV-3  was paralleled with the control group . For each treatment groups, three replicates have been done. In each tank (1.6 m × 1.2 m × 0.6 m), which contained around 1 m3 water, healthy juvenile common carp  were used. The water temperature during the experiment was maintained at 25 ± 1°C.German mirror carp selection Go CyHV-3 . Two G4  the gut , 25, 27.2.1-2011 . The CyHhttps://imagej.en.softonic.com/). In detail, the area of both trunk kidney and trunk regions were selected and measured, and the percentages between them were calculated based on eight survivors of breeding or non-breeding strain. T-tests were applied to test the significance of the difference.A selection of common carp, from either the breeding or non-breeding strains, were sacrificed. Head kidney samples were obtained at days 0 and 7 after challenge, representing the control and survivor carp. For each tissue sample, three fish were used. For transcriptomic analysis, the samples (N = 3) were collected immediately and soaked in 10 volumes of RNAlater , for sequencing using Illumina (New England Biolabs). Further, to better sequence and generate the lncRNAs, PacBio sequencing was applied to analyze the mixture of liver, spleen, and kidney, with two sample replicates. Additionally, for the validation of transcriptomic data, the qPCR samples (N = 3) were also collected using RNAlater. The number of dead fish during the experiment was counted daily to calculate the mortality rate. Meanwhile, to analyze the degree of swelling of inner organs, the proportion of trunk kidney to the whole trunk area was calculated by ImageJ , according to the instructions provided by the manufacturer. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer . RNA concentration was measured using the Qubit RNA Assay Kit in a Qubit 2.0 Flurometer . RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system .Optimized RNA-Seq strategies, including both PacBio and Illumina (New England Biolabs) sequencing , were usThe illustration of transcripts obtained from the results of PacBio could provide reference transcriptional sequences for the assembly of Illumine sequencing data, to improve sequencing quality. Therefore, Illumina (New England Biolabs) sequencing was performed on all head kidney samples. The procedure used for the preparation of the gene library and sequencing of the transcriptome followed previously published methods . Brieflyhttps://asia.ensembl.org/Cyprinus_carpio_german_mirror/Info/Index) (vs. controls in the breeding strain) and SvC-N (survivors vs. controls in the non-breeding strain), inter-strain comparison groups SvS-BvN-D7 (the comparison between survivors at day 7 for the two strains to compare survivors from the breeding to non-breeding strain) and CvC-BvN-D0 (the comparison between control fish at day 0 for the two strains to compare controls from the breeding to non-breeding strain), and group SvI-B&N. The vital genes for survival were investigated through group SvI-B&N, in which the DEGs with no significant differences (p >0.05) were compared between the survivors from the two strains with the transcripts from fish with severe illness. The revealed DEGs were annotated by GO terms and KEGG pathways, following a previously published protocol (http://geneontology.org/) and KEGG (https://www.kegg.jp/kegg/pathway.html). Enrichment of the KEGG pathways was carried out for both upregulated and downregulated genes for all comparison groups. Then to demonstrate the immune DEGs involved pathways more clearly, the gene list of the current construct common carp immune gene library was used as follows. In addition, the LncRNAs were also annotated by GO terms.All reads in the transcriptome data were mapped on the common carp genome (o/Index)  for annoprotocol , 32. In The common carp immune library was constructed by following our previously published method, which was applied to grass carp  and tilap values were adjusted using the Benjamini and Hochberg approach for controlling the false discovery rate. Genes with an adjusted p <0.05 found by DESeq2 were assigned as being differentially expressed. The following bioinformatics analysis was performed to select immune-related transcripts from the common carp immune gene library and construct the bar plots for the major immune processes and immune categories. The t-test was used to assess differences, with a false discovery rate adjusted p <0.05. Qualitative comparisons were performed between samples by counting the number of DEG. The data were rearranged in Microsoft EXCEL, and applied to plot charts by ggplot2 (2.2.1) using R language.The DEG was generated by comparing the RPKM  using the DESeq2 R package (1.16.1). The resulting p values of enriched GO terms were produced from the Kolmogorov–Smirnov test. To elucidate the detailed lncRNA-mRNA network, the immune-related GO term containing module was selected, and the relationship of involved top 100 transcripts and related lncRNAs were shown by the cystoscope software. In addition, the immune DETs  involved in the immune-related module were classified into comparison groups SvC-B, SvC-N and SvS-BvN-D7, to reveal the LncRNA modulation of immunity at the aspect of surviving from CyHV-3 infection.The weighted gene co-expression network analysis (WGCNA) was performed using the R package “WGCNA” . SpecifiThe mRNA samples used for transcriptome sequencing were also subjected to qPCR validation (n = 3), using SYBR Green PCR master mix (Bio-Rad) and CFX real-time PCR detection system (Bio-Rad), following a previously published protocol , 38. AftTK and Sph), in randomly sampled severely sick fish (N = 10) at day 7 after challenge (p <0.01). The number of dead fish for each day is listed in p <0.01) . The gene ID, annotation, primers, and fold-change information are listed in Sequencing was performed with both the PacBio RS II and Illumina platforms to analyze the gene information of the common carp. Through PacBio sequencing, 19.49 G data were obtained. Of the 258,346 ccs, 79.79% were full-length sequences, and 17,769 polished high-quality isoforms were also revealed (p <0.01) were in the largest number (p <0.01) includes “Pre-autophagosomal structure membrane” in group SvC-B, SvC-N, CvC-BvN-D0 and SvI-B&N, and the MF terms (p <0.01) includes “NF-KappaB binding” and “lysozyme activity” in group SvC-N; “cytokine activity” in group SvS-BvN-D7; “Lysozyme activity”, “MAP kinase activity” and “ubiquitin-like protein binding” in group CvC-BvN-D0.According to the enriched general GO terms Figure 3Compared with group SvC-B, there were markedly more genes involved in immune and related pathways in group SvC-N. Current revealed immune pathways in group SvC-B were “phagosome”, “regulation of autophagy”, “ubiquitin mediated proteolysis”, and “plant-pathogen interaction”  in fish  protein  were fouof Foxp3 , and hasof Foxp3 . Thus, tof Foxp3 , as wellof Foxp3 . This inof Foxp3 . For theof Foxp3 , 69. Dowof Foxp3 , and HIFof Foxp3 .Furthermore, the DEGs generated by comparing the survivors from the two strains in comparison group SvS-BvN-D7, which can provide clues for how to develop the resistance to CyHV-3 infection. The BS-advantage semaphorin, which was also regulated by LncRNA, is related to immunoregulation , 73. Thi+ T cells upon herpes virus infection, as previously revealed in the resistant strain R3  indicated a stronger ability to form the inflammasome in the breeding strain. The higher levels of NLR family CARD domain containing 3 (NLRC3), which is a negative regulator of the DNA sensor STING, suggest less sensitivity to the DNA virus in the breeding strain. The higher levels of MHC I indicate greater potential to activate response by cytotoxic CD8train R3 . For thetrain R3 , respecttrain R3 , 82. Uprtrain R3 , also sutrain R3 . Howevertrain R3  As the strain R3 , may be train R3 , and inttrain R3 . These fThe revealed regulation of resistance to CyHV-3 by lncRNA involved numerous biological processes. Among the revealed GO terms in comparison group SvC-N, “DNA replication” could indicate virus proliferation in the non-breeding strain. In survivors from the non-breeding strain, the lncRNA-regulated genes were mainly involved in innate immune cell function  and cell apoptosis. Nramp and PAI were found regulated by LncRNA in comparison groups SvC-N (up) and SvS-BvN-D7 (down). This finding indicates the more expression of Nramp and PAI in the survivors of non-breeding strain, could facilitate virus infection and proliferation for infected cell respectively, since that Nramp may serve as a virus receptor , meanwhiTherefore, based on the present findings, a hypothesis has been proposed for the immune mechanisms involved in both healthy controls and survivors from infection in both strains Figure 7The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by The Committee for the Welfare and Ethics of Laboratory Animals, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences.LS and ZJ conceived the project and designed the experiments. NW and ZJ wrote the manuscript. ZJ performed the experiments. ZJ and JXS conducted the CyHV-3 infection experiment and collected samples. CL, XH, and YG performed fish propagation and culture. XJ conducted the RT-PCR experiment. NW and X-QX coordinated the data analysis tasks. NW, HL, MS, WY, YT, JWS, and YC analyzed the data. The manuscript was revised and approved by X-QX and LS. All authors contributed to the article and approved the submitted version.This work was funded by grants from the National Key R&D Program of China (2018YFD0900302-6), The Natural Science Foundation of Heilongjiang Province (TD2019C004), China Aquaculture Research System (CARS-45-06), and Central Public-interest Scientific Institutional Basal Research Fund (2020TD31).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


On this basis, we artificially engineered human mesenchymal stem cells (MSCs) to generate exosomes with high miR-125a expression. Treatment with these miR-125a-over-expressing exosomes attenuated the migration and VM formation in nasopharyngeal carcinoma cells. In addition, the inhibitory role of these exosomes on VM formation and migration in nasopharyngeal carcinoma was also confirmed in vivo. Overall, the current study shows that MSCs can be utilized to generate exosomes with high miR-125a level, which could be therapeutic nanoparticles targeting VM formation in nasopharyngeal carcinoma and used as a complement to anti-angiogenic therapy in the future.Vasculogenic mimicry (VM) is a kind of tumor vasculature providing blood supply for tumor growth, and the formation of VM is independent of vascular endothelial cells. Instead, VM structures are formed by differentiated tumor cells such as nasopharyngeal carcinoma cells. Recently, studies have shown that anti-angiogenic therapy failed to improve the overall survival for patients, namely, nasopharyngeal carcinoma patients. The existence of VM structure is probably one of the reasons for resistance for anti-angiogenic therapy. Therefore, it is important to study the mechanism for VM formation in nasopharyngeal carcinoma. In this study, the bioinformatic analysis revealed that microRNA-125a-3p (miR-125a) was highly expressed in normal nasopharyngeal epithelial tissue than in nasopharyngeal carcinoma. An  Nasopharyngeal carcinoma (NPC) is a unique class of head and neck malignant tumor, and is endemic in eastern parts of Asia and North Africa , 2. StanAngiogenesis is a hallmark of cancer which is essential for growth, invasion, and metastases of tumors, and has long been proposed as a therapeutic target in oncology . StudiesVasculogenic mimicry is the channel conducting blood or other liquid formed by genetically dysregulated tumor cells rather than endothelial cells (ECs). Periodic acid-Schiff (PAS) staining combining with CD34 immunohistochemistry (IHC) can be used to detect the structure of VM . VM has MicroRNAs (miRs) are small noncoding, single-stranded RNAs, which are now known to play an vital role in tumor malignancy , 13. MiRExosomes are membrane-bound vesicles which can be secreted by different cells. The diameter of exosomes is reported to be 30–150 nm. Exosomes are found to be important messengers for cell–cell communication  transfervia inhibiting TAZ. Furthermore, we confirmed that MSC-exosomes could be engineered and used as anti-tumor vehicles containing miR-125a to inhibit invasion, migration, and VM formation in NPC cells in vivo and in vitro.In this study, we report for the first time that miR-125a inhibits invasion, migration and VM formation in NPCs via Mx-3000P Quantitative PCR System (Stratagene). GAPDH and U6 were selected as control group. The following were the primer sequences for different genes: miR-125a-5p forward, 5′-GGTCA TTCCCTGAGACCCTTTAAC-3′; reverse, 5′-GTGCAGG GTCCGAGGT-3′. TAZ forward, 5′-ACCCACCCACGATGACCCCA-3′; reverse, 5′-GCACCCTAACCCCAGGCCAC-3′; GAPDH, forward, 5′-GGAGCCAAAAGGGTCATCAT-3′; reverse, 5′-GTGATGGCATGGACTGTGGT-3′. U6 forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse 5′-AACGCTTCACGATTTGCGT-3′. The relative mRNA level was normalized by the expression of GAPDH, and the relative miR level normalized by expression of U6. The independent experiments were performed 3 times, each of which used 3 samples independently.Trizol reagent  was used to extract the total RNA in NPC cell lines. Then the reverse transcription was continued referring to the protocol by High Capacity cDNA Reverse Transcription Kit (Biosystems). The cDNA was utilized for real-time PCR http://www.targetscan.org/), miRbase (http://www.mirbase.org/) and miRDB (http://mirdb.org/miRDB). The scores and strength of binding sites for predicted target were detected. The miRNAs overlapping in more than 2 searches were selected for further studies.The TAZ gene was queried by the miRNA target prediction sites TargetScan (Human) (https://tcga-data.nci.nih.gov/tcga/). All the data were analyzed with GraphPad Prism.The clinical information and also microRNA expression levels for clinical samples in NPC and HNSCC were acquired from the GEO (Gene Expression Omnibus) database (NCBI/GEO/GSE32960) and TCGA database  with 10% FBS, supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. The keratinocyte serum-free medium  with human recombinant epidermal growth factor (rEGF) was used to culture the NP-69 cells and NP-460 cells which were immortalized nasopharyngeal epithelial cell line. The 293 T cells were maintained in our lab and were cultured for luciferase reporter assay.MSC were cultured and the exosomes were isolated from the conditioned medium using Exosome isolation Kit—Serum and Plasma (Exiqon) according to protocol, the procedures of which were performed in previous studies . The eleThe pGL3-TAZ and pGL3-mutTAZ reporter genes were both established by Bio-Asia (China). The luciferase reporters and the miR-125a mimics were both transfected in 293T cells. The activity of the reporter protein was detected by a luciferase assay kit (Promega) after transfected for 48 h with reference to the instructions.The paraffin-embedded tissues were sliced and heated for further investigation. After deparaffinized, rehydrated and antigen retrieving, the slides were blocked with goat serum and incubated with primary antibody (Abcam) at 25°C for 4 h. After the usage of a secondary antibody and DAB  substrate solution, the picture were captured. Matrix-associated vascular channels including the vasculogenic mimicry structure were detected by PAS staining. Images for all the slides were captured by Leica DM 2500 microscope.4 cells per well) and cultured for 18 h by RPMI 1640 medium without FBS. Images were took after 36 h using Leica DM 2500 microscope randomly.Matrigel were coated in 96 wells plates (50 μl/well), and NPC cells (6-10B and 5-8F) were seeded in the wells . Lentiviruses expressing miR-125a, inhibiting miR-125a and the scrambled control were constructed by Genechem . The transfecting with lentivirus were performed at MOI = 10 in the NPC cells and at MOI = 10 in the human mesenchymal stem cells.NPC cells were seeded in the top chamber cultured by serum-free media. The bottom chamber was filled with RPMI 1640 medium containing 15% FBS. After 36 h (6–10 B) or 24 h (5–8 F) of incubation, the cells were simply removed and fixed with 4% paraformaldehyde in the top chamber for 20 min, and then stained with giemsa solution for 20 min. Five fields per well were photographed at random.2 and then the tumor growth curves were generated.A total of six BALB/c nude mice  were purchased in the SLAC Laboratory Animals (Shanghai), and were randomly divided into 2 groups. After disinfection, stably transfected cells  were subcutaneously injected at the left groin. Tumor growth was detected, and the diameters of long (a) and short (b) for each tumor were measured every 3 days. Tumor volumes were calculated as V = 0.5 × a × bvia ImageJ 1.52a software  and normalized to the bands of GAPDH.The cultured cells were harvested using RIPA cell lysis buffer. All the protein lysates in different groups were loaded and separated by SDS-PAGE, and the SDS PAGE gel were transferred to a polyvinylidene difluoride (PVDF) membrane. The blots were incubated with primary antibodies against TAZ , LAMB2 , N-cadherin , E-cadherin , MMP2 , MMP9 , and GAPDH . The enhanced chemiluminescence  was utilized for visualizing the protein bands. The intensity of the protein bands was analysed NPC cells were cultured in RPMI 1640 medium containing 15% FBS in 6-well plates at 37 °C for 24 h. Wounds were generated by scratching with 200 ul sterile pipet tips, and washed 3 times with phosphate-buffered saline (PBS). After wounds were made, cells were cultured in RPMI 1640 medium containing 1% FBS. Then images were captured at the time 0 h and 36 h after wounding.via log-rank tests using GraphPad Prism 7.00 software. All tests were two-sided, and p-values <0.05 were considered statistically significant. All data are presented as the mean ± standard error of the mean. Bar graphs represent mean and SD or mean and SEM across three independent experiments.All statistical data were analyzed using GraphPad Prism 7.00 software. Student’s t-test was used for calculating significance between two groups, and one-way ANOVA was used for multiple comparisons. Kaplan–Meier survival curves were also analyzed ). In addition, patients with high expression of mir-125a had higher survival rate as well as better prognosis  database (NCBI/GEO/GSE32960). Heatmap  FigureFigure. ITo sum up, these results indicated that the low level of miR-125a is related to development of NPC cells.We showed VM structures in NPC tissue by double staining with PAS and CD34, in line with previous studies in other neoplasms. VM structures could be observed in NPC tissues, which were negative for vascular endothelial marker CD34 but positive for PAS  and EMT-related protein level  , we therhttp://www.targetscan.org/), miRwalk , miRDB (http://mirdb.org/) and the TCGA database for prediction, and 25 genes were selected  (We subsequently identified the candidate target-genes according to the following criteria: (1) metastasis-related genes in NPC based on LinkedOmics (cs.org/) ; and (2)).Kaplan–Meier survival curves in HNSCC were constructed using the TCGA clinical data, and low TAZ level were prognostic of better overall survival in HNSCC patients . The lucFurthermore, the effects of the knockdown of TAZ resembled the effects of miR-125a overexpression, which decreased the protein levels of E-cadherin, LAMB, MMP9, and N-cadherin , the Science and Technology Commission of Shanghai Municipality (project No. 19411951000), the Science and Technology Development Fund of Shanghai Pudong New Area (No. PKJ2020-Y53) and the Shanghai Sailing Program (No. 20YF1428000).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Health literacy is considered to be an emerging determinant of health behaviors and outcomes. The underlying mechanisms linking health literacy to diabetes self-management are currently unclear. This study assessed a mediation model consisting of a direct pathway between health literacy and self-management, and indirect pathways via social isolation only, self-efficacy only, and social isolation and self-efficacy serially in people with type 2 diabetes. A cross-sectional design was employed, and a total of 524 participants were recruited from outpatient clinics of multi-institutions from June 2020 to February 2021. The mediation model was analyzed using the PROCESS macro on SPSS with bootstrap bias-corrected 95% confidence intervals (CIs) with 10,000 bootstrapping iterations. Health literacy positively affected self-management. The estimated indirect effect of health literacy on self-management via social isolation was significant, at 0.018 (95% CI = 0.004–0.036). The indirect effect via self-efficacy was estimated at 0.214 (95% CI = 0.165–0.266). The indirect effect via social isolation and self-efficacy serially was 0.013 (95% CI = 0.006–0.023). The findings of this study suggest that clinical practice can be improved through more comprehensive diabetes self-management interventions that promote all of the components of health literacy, social contacts/networks, and self-efficacy in particular. Diabetes is a global health problem. Approximately 463 million people had diabetes worldwide in 2019, and this number is estimated to reach 700 million by 2045 [Health literacy has been described as “cognitive and social skills that determine the motivation and ability of individuals to gain access to, understand, and use health information in ways that promote and maintain good health” . Health According to social cognitive theory, self-efficacy is “the belief in one’s capability to organize and execute the course of action required to produce given levels of attainments” . Self-efSquiers et al.  suggesteSocial isolation may be a plausible mediator, which is a structural indicator of social connection that refers to an objective and quantitative measure of network size, diversity, and interpersonal contact frequency . Social People with lower health literacy levels tended to be less interested in social engagement , and greSocial isolation was reported to be significantly correlated with self-efficacy in people with type 2 diabetes . Wu and Based on the literature review, the present study assessed a mediation model for people with type 2 diabetes that consisted of the following hypothesized pathways: a direct effect of health literacy on self-management, and three different indirect (mediation) effects via social isolation only, self-efficacy only, and both social isolation and self-efficacy serially.This study had a cross-sectional design. A total of 524 participants were recruited from outpatient clinics of multiple institutes in the Republic of Korea from June 2020 to February 2021 using convenience sampling. The sample size satisfied the mediation model recommendation of 450–500 cases for a statistical power of 80% to validate indirect effects ,28. The This study was approved by the institutional review boards of the hospitals from which the participants were recruited. The participants were provided with information about the purpose of this study, the voluntary nature of participation and the right to refuse to answer or withdraw, and risks and benefits of participation. All participants signed the informed-consent form and received remuneration.The Diabetes Health Literacy Scale (DHLS) was used, which is a disease-specific health literacy instrument  that conShankar et al.  developeSelf-efficacy was determined by the four-factor, 16-item Korean version of the Diabetes Management Self-Efficacy Scale (K-DMSES)  derived Self-management was assessed using the Diabetes Self-Management Scale (DSMS), which is a new instrument that measures the complex nature of self-management among people with type 2 diabetes, and consists of 17 items scored on a five-point Likert scale . The DSMQuestionnaires on gender, age, monthly income, education status, and diabetes education were administered. Diabetes education was assessed using the following yes/no question: “Have you ever received education about diabetes?” Medical information on treatment regimens, glycated hemoglobin (HbA1c) level, and the duration of diabetes were collected from the medical records of the participants.t-test, ANOVA, or Pearson’s correlation analysis from the demographic and medical information. The hypothesized mediation model was analyzed using the PROCESS macro with 10,000 bootstrap bias-corrected 95% confidence intervals (CIs). If a 95% bootstrap CI of a mediation (indirect) effect did not contain zero, it was considered statistically significant [Data were analyzed using the PROCESS macro (version 3.5.3) of SPSS (version 25.0 for Windows). The general characteristics of participants and study variables were analyzed using descriptive statistics. Pearson’s correlation analysis was used to identify correlations between the study variables. A covariate (confounding variable) related to both mediator and dependent variables may affect mediation effects . Possiblnificant .The mean age of the participants was 60.35 years (SD = 11.19 years). The participants included 64.1% males, 39.4% high-school graduates, 46.9% who earned more than 3,000,000 KRW monthly, and 75.6% who were taking oral hypoglycemic agents as a treatment regimen. Controlled blood glycemia levels (HbA1c < 6.5%) were observed in 25.4%, and the mean diabetes duration was 11.77 years (SD = 8.72 years). Diabetes education had not been experienced by 24.4% of the participants. Significant correlations were found between health literacy, self-efficacy, and self-management. Social isolation was negatively correlated with all of the other study variables .t = –3.03, p = 0.003)  and self-management . Diabetes education was therefore controlled as a covariate during the mediation analysis. Other demographic and medical information were not significantly associated with both mediation variables  and self-management.Participants who had received diabetes education demonstrated significantly lower scores for social isolation (= 0.003)  and highc) of health literacy on self-management was divided into direct and indirect components. The direct effect (c’) was the influence of health literacy on self-management while controlling for all other variables. The three indirect effects were the influence of health literacy on self-management via social isolation only (a1b1), self-efficacy only (a2b2), and both social isolation and self-efficacy serially (a1db2).When controlling for diabetes education for the mediation model , the totc = 0.516, 95% bootstrap CI = 0.453–0.580) and direct  effects of health literacy on self-management were positive and significant, as the CI did not include zero. The mediation model of a1b1 (–0.275 × –0.067) = 0.018, with a 95% bootstrap CI of 0.004–0.036. This indirect effect was significant, indicating that social isolation mediated health literacy and self-management while controlling for self-efficacy and diabetes education. The second indirect effect of health literacy on self-management via only self-efficacy while controlling for social isolation and diabetes education was significant, and estimated as a2b2 (19.521 × 0.011) = 0.214, with a 95% bootstrap CI of 0.165–0.266. Those with greater health literacy therefore had greater self-efficacy, which induces greater self-management, and was independent of social isolation and diabetes education. The third indirect effect of health literacy on self-management via social isolation and self-efficacy serially while controlling for diabetes education was estimated as a1db2 (–0.275 × –4.425 × 0.011) = 0.013, with a 95% bootstrap CI of 0.006–0.023, indicating that the indirect effect from these two serial mediators was significant while controlling for diabetes education. The total sum of the indirect effects was also significant, and was estimated as a1b1 + a2b2 + a1db2 (0.018 + 0.214 + 0.013) = 0.245, with a 95% bootstrap CI of 0.196–0.299  . HoweverThe third strength of the present study was the type of analysis used to determine mediation effects. The mediation model in this study was assessed using the PROCESS macro with bootstrapping to account for the inferential limitations when assuming a normal distribution. This approach had greater statistical power than that proposed by Baron and Kenny , and theA systematic review of empirical studies on the pathway linking health literacy to health behaviors and outcomes suggested that most studies have not controlled for covariates . The fouNotwithstanding the abovementioned strengths, the findings of this study must be cautiously interpreted due to its cross-sectional design, where temporal sequence in the mediation model was difficult to identify. It is therefore suggested to test the mediation model using a longitudinal research design in the future.This study demonstrated that health literacy affected diabetes self-management via one direct and three specific-indirect pathways through social isolation only, self-efficacy only, and both social isolation and self-efficacy serially. The magnitude of the indirect effect via self-efficacy was larger than those of the other indirect effects. The mediation model indicated that clinical practice can be improved by providing a more comprehensive self-management intervention to people with diabetes that enhances all the components of health literacy, social contacts/networks, and self-efficacy in particular.


The World Health Organization declared COVID-19, the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a global pandemic on March 11, 2020. Non-pharmaceutical interventions such as social distancing, handwashing, using hand sanitizer, and wearing facial masks are recommended as the first line of protection against COVID-19. Encouraging hand hygiene may be one of the most cost-effective means of reducing the global burden of disease.This study uses a web-based questionnaire to evaluate the usage patterns and consumer perceptions of the effectiveness and health safety of bar soap, liquid hand soap, and hand sanitizer products before and after the spread of COVID-19.The results show that since the outbreak of COVID-19, the number of consumers who primarily use bar soap has decreased from 71.8 to 51.4%, the number of those who primarily use liquid hand soap has increased from 23.5 to 41.3%, and the number of those who use and carry hand sanitizer has increased. The frequency of use, duration of use, and amount used of all three products have increased significantly since the COVID-19 outbreak. Finally, consumer perception of the products’ preventive effect against COVID-19 is higher for liquid hand soap and hand sanitizer than it is for bar soap.Because use of hand sanitizers has increased, public health guidelines must address the potential risks associated them. Our data also show that the public is abiding by the recommendations of the regulatory authorities. As handwashing has become important in preventing COVID-19 infections, the results of our study will support the development of better handwashing guidelines and a public health campaign.The online version contains supplementary material available at 10.1186/s12302-021-00517-8. Handwashing habits were evaluated before and after the spread of COVID-19.Handwashing habits increased significantly since the COVID-19 outbreak.Handwashing guidelines and campaigns be developed informing the potential risks.The online version contains supplementary material available at 10.1186/s12302-021-00517-8. A number of pneumonia cases, in which the cause was unknown, appeared in Wuhan, Hubei Province, China, in December 2019 . The numCOVID-19 can be transmitted through droplets and contact. It is not yet clear whether COVID-19 can be transmitted through aerosols containing the virus, including absorption of the virus through the digestive tract . ContamiWith no available drugs or vaccines, non-pharmaceutical interventions such as social distancing, handwashing, using hand sanitizer, and wearing a face mask are recommended as the first line of protection against COVID-19 , 8. A rePrevious studies have suggested that encouraging hand hygiene may be one of the most cost-effective means of reducing the global burden of disease . HoweverIn this study, we analyzed changes in handwashing patterns before and after the pandemic to provide better handwashing guidelines and to prepare for future public health crises. Our research on changes in handwashing behavior could contribute to providing consumers with the appropriate handwashing guidelines and developing better strategies for a public campaign.A total of 1000 Korean consumers participated in this study, and the survey was conducted between July 27 and August 19, 2020. Respondents were appropriately distributed by gender and age group .A web-based questionnaire was used to evaluate the usage patterns and perceptions of bar soap, liquid hand soap, and hand sanitizer before and after the spread of COVID-19. The questionnaire first asked about participants’ handwashing habits, including questions about which products they use, the amount of product they use, the duration of each, if they have any concerns about the products’ safety, and their perception that the product can prevent against COVID-19 infection. To determine handwashing patterns before the COVID-19 pandemic, participants were asked to answer a series of questions based on their habits one month prior to the outbreak. For each product group, questions about participants’ safety concerns and their perception of the product’s ability to prevent the spread of COVID-19 were asked on a 7-point scale. The questionnaire was conducted in Korean, but an English translation is provided in the Additional file p-value < 0.05 was considered significant.Data were analyzed using RStudio version 1.3.1073  and SPSS version 21.0 software . A The demographic breakdown of the questionnaire respondents is 50.9% male and 49.1% female, which is representative of the national population . We constructed five age groups: 20s (20–29 years), 30s (30–39 years), 40s (40–49 years), 50s (50–59 years), and 60s (60–69 years). These groups contained 19.4%, 19.9%, 23.5%, 24.4%, and 12.8% of the respondents, respectively. The group with the highest bias  was the 60s (–2.9%)  in the products used before and after the outbreak of COVID-19  for handwashing in Korea (1000 consumers were surveyed) pre- and post-COVID-19 were investigated and compared with Crème data , a studyp-value < 2.2E−16, and a significant change was detected between pre- and post-COVID-19 use. The number of consumers using hand sanitizer increased from 14.6% pre-COVID-19 to 89.8% post-COVID-19  did not use hand sanitizer before the COVID-19 outbreak. The peak in frequency of use was once per day, the duration of use was 0 s, and number of pumps was zero. The second-highest peak for the duration of use and number of pumps used for hand sanitizer was 10 s and one pump, respectively. After the outbreak, the peak was three times per day, 10 s duration, and one pump Fig. .Fig. 2CoWe analyzed how many times during the day the respondents washed their hands and how long it takes for them to wash only their hands. The analysis excludes those who responded “no use”. The average frequency of handwashing per day increased from 6.96 times to 9.98 times after the COVID-19 outbreak. Similarly, the average time taken to wash one’s hands increased from 20.16 to 31.58 s Table . AccordiIt was found that the highest proportion of Korean consumers (20% and 23.3%) washed their hands five times a day pre-COVID-19, and they wash 11–20 times a day post-COVID-19. According to Crème data , 21% of p-values of the t-test are presented in Table F-value = 1.0E−4). As a result of the t-test for the two products, bar and liquid hand soap differed in average value with a p-value of 1.15E−4. Bar soap and hand sanitizer also showed a difference in average value with a p-value of 4.693E−4. However, the perceived preventive effect of liquid hand soap and hand sanitizer did not differ . The perceived preventive effect against COVID-19 was higher for liquid hand soap and hand sanitizer than it was for bar soap.The latter part of the questionnaire asked the respondents about their use of hand-washing products before and after the COVID-19 outbreak . The mean, standard deviation, and In the wake of the COVID-19 outbreak, frequent handwashing and proper hand hygiene are strongly recommended by international health agencies and Korean regulatory agencies, such as the Korean Center for Disease Control and the Ministry of Food and Drug Safety, as the easiest, most economic, and most effective ways to prevent the spread of the virus. The Centers for Disease Control and Prevention (CDC) recommends washing hands with soap and water for at least 20 s or using a hand sanitizer with at least 60% alcohol to prevent the spread of germs during the COVID-19 pandemic . The hanA total of 1123 participants were dropped from the survey, either because they did not complete it or responded “no wash”. The results do not distinguish between those who responded “no wash” and those who did not complete the survey. Therefore, at most, 52% 1123/2123) of the survey respondents do not wash their hands. A 2014 study by Freeman et al.  may have23 of theThe number of Korean consumers who use liquid hand soap has increased (from 23.5% before the outbreak to 41.3% after). Although, many consumers still use bar soap (51.4%), the number of consumers using only bar soap decreased significantly from 41.4% before the pandemic to 17% after .The use of hand sanitizers containing biocidal alcohol  among Korean consumers increased from 14.6 to 89.8%. In addition, the number of consumers carrying hand sanitizer increased from 4.1 to 39.3%. This change is due to consumers’ perception that hand sanitizers are more effective at killing the virus that causes COVID-19 than handwashing with soap. By contrast, In Korea, humidifier disinfectants containing biocidal ingredients caused 6246 reported cases of and 1375 deaths by pulmonary fibrosis as of February 28, 2018 -28. ThesIn the present study, we examine Korean behavior patterns related to bar soap, liquid hand soap, and hand sanitizer before and after the COVID-19 outbreak, as well as consumer concerns regarding the safety of the three products studied and consumer perception of the products’ effectiveness in preventing the spread of COVID-19. The data collected not only provide insight into consumer behavior patterns, such as the frequency of use, the duration of use, and the amount of product used, but they also show how these patterns have changed since the COVID-19 outbreak. As consumer use of hand sanitizers has increased, public health guidelines must address the potential risks associated with them. Our data also show that the consumers are abiding by the recommendations of the regulatory authorities for handwashing. As handwashing has become important in preventing COVID-19 infections, our analysis of changes in handwashing behavior that occurred after the COVID-19 outbreak will support the development of better handwashing guidelines and a public health campaign.Additional file 1: Questions for selecting respondentsAdditional file 2: Table S1. Comparison of bar soap, liquid hand soap and hand sanitizer usage patterns of all respondents pre- and post-COVID-19. Table S2. Ratio of co-use of bar soap, liquid hand soap and hand sanitizer before and after the outbreak of COVID-19 by age. Table S3. Ratio of co-use of bar soap, liquid hand soap and hand sanitizer before and after the outbreak of COVID-19 by occupation. Table S4. Comparison of average handwashing frequency and average handwashing duration pre- and post-COVID-19 by age. Table S5. Comparison of average handwashing frequency and average handwashing duration pre- and post-COVID-19 by occupation. Table S6. Comparison of bar soap, liquid hand soap and hand sanitizer usage patterns of consumers using products pre- and post-COVID-19.


Myasthenia gravis (MG) is an auto-immune disease, and the mainstay of therapy is immunomodulation. Such patients are at high risk of acquiring any infections. Hence, we sought to determine the impact of the current global pandemic COVID-19 infection in MG patients.TM that was provided through Cerner’s HealtheDataLab research tool. We ran a database query from January 2019 to July 2020 in our study and identified myasthenia patients with and without COVID-19 infection. To extract these patients’ data, we used ICD 9-CM, ICD-10, and SNOMED-CT codes. We reported the data using means, range, and prevalence rates, and the p-values were calculated using the two-sample t-test and Pearson’s chi-squared test.For our study, we used Cerner Real-World Datan = 7), and the non-COVID-19 set was 4.33 days (n = 6), and it was statistically significant .In the COVID-19 data set, a total of twenty-seven myasthenia patients were identified with a positive COVID-19 infection, and four were diagnosed with an exacerbation. The male to female ratio was equal and one unknown gender (3.7%) with a mean (± SD) age of 64.33 ± 18.42 years. This study group was compared with a non-COVID-19 data set in which a total of sixty-four myasthenia patients were identified, and twenty-three had an exacerbation. Among the 13 hospitalized patients in the two groups, the mean length of hospitalization for the myasthenia patients in the COVID-19 data set was 8.28 days (The mean length of hospital stay is prolonged in myasthenia patients who tested positive for COVID-19. Myasthenia Gravis is an auto-immune disease characterized by fluctuating weakness in the voluntary muscles caused by antibodies towards the neuromuscular junction. When severe weakness develops in the respiratory muscles, it leads to a significant life-threatening complication called myasthenic crisis, resulting in respiratory failure . Around TM provided through Cerner’s HealtheDataLab research tool  COVID-19 Associated Risks and Effects in Myasthenia Gravis, a physician-reported registry is currently open and active . In addiOur study utilized the de-identified real-world data from the CERNER database, and the data was analyzed to report the characteristics of the patients and the outcomes. Comorbidities like hypertension, diabetes mellitus, cardiovascular disease, and respiratory diseases may pose a significant risk of developing complications in patients with COVID-19 infection , 12. In Infections are the most common triggering factors to develop exacerbation in myasthenia patients , 14. AcuA single case of myasthenic crisis secondary to COVID-19 infection was reported [a statistically significant higher mean length of hospitalization of 8.28 days in the COVID-19 group among the hospitalized patients as compared to 4.33 days in the non-COVID-19 group. In an observational study, the average length of hospitalization from the myasthenia exacerbation was reported as 6.5 days [Among the 27 COVID-19 positive myasthenia patients, seven patients were hospitalized, and the calculated mean length of stay is 8.28 days. The mean length of hospitalization in the non-COVID-19 group was 4.33 days. Only 6 out of 64 myasthenia patients were hospitalized in the non-COVID-19 group. Our study observed 6.5 days . In a la6.5 days .The mean length of hospital stay is prolonged in myasthenia patients who tested positive for COVID-19.The study protocols were approved by the Institutional review of Board committee at University of Missouri Columbia. All methods were carried out in accordance with relevant guidelines and regulations of the committee. The ethic committee waived the informed consent as it is de-identified data. The limitations of our study are that we lack the availability of the data publicly. However, in all the centers located in the US, we lack demographical localization of the health centers and lack of seropositivity results in all myasthenia patients.


Dexamethasone-loaded polymer hybrid nanoparticles were developed as a potential tool to treat alopecia areata due to their follicular targeting ability. Freeze drying (FD) is a common technique used to improve nanoparticle stability; however, there are few studies focused on its effect on ethyl cellulose lipid-core nanoparticles. Nanoparticles were lyophilized with different cryoprotectants. Sucrose was selected because it allowed for a good resuspension and provided acceptable physicochemical parameters . The nanoparticles obtained were loaded into a pleasant xanthan gum hydrogel, and the rheological, release, and skin permeation profiles of different formulations were studied. The FD formulation significantly modified the particle size, and the drug release and permeation properties were also altered. In addition, analyses of the cytotoxicity and anti-inflammatory efficacy of FD and non-FD particles on human keratinocytes indicated no differences. Nanoparticles have a high surface-to-volume ratio, which is very interesting for topical applications as they increase skin–nanoparticle contact and improve drug diffusion into the skin . In addiAlopecia areata is an inflammatory disease that affects between 1% and 2% of the general population . It invoPolymeric lipid hybrid nanoparticles, also called lipomers, are promising vehicles for the topical administration of hydrophobic drugs. The lipomers contain a polymeric matrix, which can control drug release by diffusion, erosion, or swelling (depending on the polymer type used) and lipids, which are able to increase the loading capacity of hydrophobic drugs. These particles are stabilized by amphiphilic compounds on the nanoparticle surface. Nanoparticle suspensions could have different instability issues, such as drug leakage in aqueous medium, particle aggregation, polymer hydrolysis, issues related with the chemical stability of the entrapped drug , etc. A The aim of this research was to study the impact of the FD process (with different cryoprotectants) of ethyl cellulose DEX-lipomers on the physicochemical, biopharmaceutical (drug release and ex vivo skin permeation), and in vitro anti-inflammatory properties (on human keratinocytes) of the obtained particles. Nanoparticles were produced with ethyl cellulose (EC)  and medium-chain triglycerides (MCT) ; Tween 80 and Span 60  were used as stabilizers, and benzalkonium chloride  was chosen as preservative. DEX  was the active pharmaceutical ingredient, and the selected solvents were ethyl acetate (EA), ethanol absolute (ET) , and purified water (Inhouse). The cryoprotectants used in the freeze-drying process were sucrose , trehalose, and mannitol . w/w, 0.2% w/w, 0.16%, and 1% w/w, respectively) were dissolved in EA:ET (5:1) and emulsified with purified water with tween 80 and the preservative benzalkonium chloride  with a UP400st ultrasonic device  with an amplitude of 40% for 5 min. The organic solvent of the emulsion was evaporated under vacuum at 40 °C for 5 min. Cryoprotectants were added after nanoparticle production. A schematic representation of the lipomers is presented in The DEX-lipomer formulation was previously developed by an emulsion solvent evaporation method . BrieflyFreeze-Drying Microscopy (FDM) was carried out to determine the temperature at which the different thermal events occur in the formulation of lipomers and thus define the optimal lyophilization cycle. The system used was an Olympus BX51 Lyophilization Microscope , a PixeLINK camera, with a liquid nitrogen cooling system, a Linkam temperature controller TMS 94, a liquid nitrogen pump , a 10× magnifications objective, a Linkam FDCS 196 Freeze-Drying Stage  attached to the Freeze-Drying Stage system, and a Pirani Gauge pressure controller .w/w) were added to the DEX-lipomers and subjected to a lyophilization cycle together with the nanoparticles without lyoprotectant to compare their effect on the reconstitution of the particles.A drop of 10 µL of freshly prepared placebo lipomers, DEX-lipomers, DEX-lipomers with 6% trehalose, DEX-lipomers with 6% sucrose, and DEX-lipomers with 6% mannitol were added, and a cooling ramp was carried out from 25 to −80 °C at a rate of 10 °C/min. The temperature was stabilized at −80 °C for 3 min, and finally, a temperature ramp was applied from −80 to −5 °C at a rate of 2 °C/min; 600 photographs were taken throughout the event. Three different lyoprotectants  was used for the lyophilizer. From the information obtained in the Lyophilization Microscope studies, the parameters of temperature, pressure, and time were established for each freeze-drying cycle.Dynamic Light Scattering (DLS) ) was used to study the Hydrodynamic size (Z-ave), Polydispersity Index (PDI), and Zeta-potential (Z-pot) of the produced nanoparticles. Prior to measurements, a 1:10 dilution of nanoparticles in milliQ water was carried out.w/w at 25 °C until samples were dried. Transmission Electron Microscopy (TEM)  was employed to study the size and morphology of the nanoparticles before and after the lyophilization process. A dilution of 1:10 non-freeze-dried (non-FD) DEX-lipomers and water resuspended FD DEX-lipomers was prepared in milliQ water, placed in TEM grids, and stained for 1 min with Uranyl Acetate solution 2% Differential Scanning Calorimetry (DSC) analyses were performed in a DSCq20  to observe the thermal events taking place in the formulations before and after the lyophilization process. Samples of 3 mg DEX, freeze-dried DEX-lipomers (FD DEX-lipomers), and non-FD DEX-lipomers were placed in Tzero Aluminum pans and sealed with Tzero hermetic aluminum lids with a Tzero press . Temperature ramps were applied from 0 to 200 °C at 10 °C/min for the formulations containing sucrose and from 20 to 277 °C at 10 °C/min for the formulations without sucrose. This was because sucrose degrades at approximately 210 °C  and, in w/w under magnetic stirring at 500 rpm for 1 h at room temperature to solutions of DEX-lipomer, placebo hydrogel, and DEX-hydrogel formulations.Xanthan Gum was added at a concentration of 0.75% −1. Formulation rheology studies were performed at 25 °C, 24 h after formulation production. Measurements were performed with a Bohlin VOR rheometer . Studies were performed with parallel plate‒plate geometry (PP30) with a 0.5 mm gap. A strain sweep test was performed with a strain range of 1–100% and a 1 Hz oscillation rate. Approximately 3.5 g was placed on the plates. The viscosity was measured with a shear rate of 0.1–6.3·sδ), viscosity (η), thixotropy, and rheological behavior. The obtained rheological behavior experimental data were fit to different equations , loss modulus (G″), delta angle , and the best fit was selected based on the lowest equation cost (Equation (7)).ix denotes the empirical shear rate, and iy denotes the empirical shear stress. Model fitting was performed with Python software  passed at 1.8 mL/min through a C18 HPLC column  at 25 °C. The detection wavelength was 208 nm, and the injection volume was 20 µL. The assay was performed with an HPLC instrument . In addition, this method was used to estimate the encapsulation efficiency (%EE) according to Equation (8), where WT denotes the total content of DEX in the formulation and NEW denotes the DEX obtained in the filtrate (not encapsulated) after centrifugation of the amicon ultra device  with a membrane cut-off of 100 KDa at 4500 rpm for 30 min.A previously described HPLC method  was empl2. Experimental variables were the same as previously used . ThAn in vitro permeation test was performed with dermatomed pig skin on fresh non-FD DEX-lipomers, FD DEX-lipomers, hydrogel-loaded FD DEX-lipomers, and free DEX hydrogel. pK or supJ. Although there were differences in the maximum permeated drug concentration, the slope of both formulations was similar, and the same transdermal flux was obtained. The significance of P1 (diffusion-related parameter) was borderline (p = 0.08), which was probably related with the differences in drug release; however, the power of the statistical test was not enough to discriminate between both formulations. Clear statistical differences were found in lag time and related parameters . The FD process increased the lag time, which was probably caused by the increase in particle size (double that of non-FD particles) and the modification of nanoparticle structure. The greater particle size can affect the number of nanoparticles that are stored in the hair follicles and may reduce the packaging of the particles in the skin surface, i.e., the greater the size, the lower the surface-to-volume ratio. The modification of the lag time brings about the modification of P2 (the partitioning-related parameter), which is reduced compared with the non-FD formulation. This could be caused by the reduced amount of intimate contact between both systems (due to the increase in particle size and the reduction in the surface-to-volume ratio), which may lead to a decrease in P2. Another possibility is that the cryoprotectant modifies the formulation/skin partitioning, because these compounds can form hydrogen bonds with the nanoparticle surface and alter the interaction with the skin.Additionally, the permeation parameters were estimated . No statHuman-transformed normal epidermal keratinocytes (HEK001) and human immortalized keratynocytes (HaCaT) were chosen to determine the cytotoxic effect of FD DEX-lipomers compared to the non-FD DEX-lipomers. Non-loaded non-FD lipomers with sucrose (sucrose) were tested as controls. HEK001 cell viability profiles exhibited the same pattern as that previously observed in non-FD DEX-lipomers  Figure A. HoweveTo ascertain the in vitro anti-inflammatory efficacy of DEX-loaded lipomers, we selected the cytokine TNFα as a marker, considering its role in most inflammatory alterations, including those related to the skin such as alopecia areata ,35. The Previously developed DEX-lipomers composed of ethyl cellulose and medium chain triglycerides were freeze-dried. The lyophilization process significantly modified the nanoparticle internal structure and size, which was probably caused by the crystallization of the oil core. This significantly modified the drug release characteristics and the permeation profile of dermatomed pig skin compared with the non-freeze-dried particles. Xanthan gum hydrogel was loaded with DEX-lipomers, which significantly changed the rheological behavior compared with the placebo (free DEX) hydrogel. Regarding the in vitro anti-TNFα analyses, no significant differences were observed between the freeze-dried and the non-freeze-dried particles. The most interesting biopharmaceutical properties were achieved with the non-freeze-dried formulation in suspension. An adequate balance between stability, efficacy, patient texture preference, and biopharmaceutical properties must be established to obtain a successful drug delivery system.